Characterisation of 96 mutations in 128 unrelated severe haemophilia A patients from France

2006 ◽  
Vol 95 (04) ◽  
pp. 593-599 ◽  
Author(s):  
Christine Vinciguerra ◽  
Christophe Zawadzki ◽  
Yesim Dargaud ◽  
Gilles Pernod ◽  
Claire Berger ◽  
...  
Keyword(s):  
Factor Viii ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Haemophilia A ◽  
Missense Mutations ◽  
Severe Haemophilia ◽  
Coding Region ◽  
Nucleotide Substitutions ◽  
Large Deletions ◽  
Fviii Inhibitor

SummaryDirect sequencing of the coding region of factor VIII (F8) gene was used to determine the mutations responsible for severe haemophilia A (FVIII<1%) in 128 unrelated haemophiliacs A, negative for intron 22 and intron 1 inversions. A mutation was found in 122/128 patients (95%). Ninety-six distinct mutations were identified in this cohort, 62 of these are novel. They consisted of deletions (7 large and 24 small deletions), insertions (n=9), associations of insertion/deletion (n=2), association of deletion/substitution (n=1), and single nucleotide substitutions (53 point mutations consisting of 31 missense, 20 nonsense, and 2 splicing mutations). Twenty-two patients had developed inhibitors, and among this subgroup 3 large deletions, 6 frameshift, 9 nonsense and 4 missense mutations were detected. For6 patients, among which one developed an anti-FVIII inhibitor, no mutations were detected in the coding and splicing regions of factor VIII gene. Different approaches of molecular modelling were performed in addition to familial linkage analysis to determine the pathophysiological responsibility of these novel missense mutations.

F VIII Inhibitors in Haemophilia A Patients who Are Considered as Previously Treated Patients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4022-4022
Author(s):  
Ch. von Auer ◽  
M. Krause ◽  
W. Miesbach ◽  
I. Scharrer ◽  
G. Asmelash ◽  
...  
Keyword(s):  
Factor Viii ◽  
Haemophilia A ◽  
Missense Mutations ◽  
Genotype 4 ◽  
Large Deletions ◽  
Factor Viii Concentrates ◽  
Previously Treated Patients ◽  
Previously Treated ◽  
Factor Concentrate ◽  
Continuous Use

Abstract Previously treated patients (PTP), who have not developed an inhibitor (inh) so far, are considered to be tolerant to factor VIII and at low risk for inh development. Therefore inh detection in a PTP should raise concerns about the concomitant variables such as product neo-antigenicity or way of application. In our own center we recently detected the new development of a high responding inh to factor VIII in a 58 year old patient with severe haemophilia A. To find out about the current situation regarding inh development in PTP in Germany, we conducted a retrospective study. A questionnaire was sent to 99 haemophilia treating physicians, so far 46 of them answered. 24 PTP-inh were registered during the last 5 years. Patients had at least 20 ED and/or one change of factor concentrate. Age (9 months to 70 years, median 35), severity of haemophilia A (16 severe, 2 moderate, 6 mild), exposure days (ED 6 to >1500, median 37) and genotype (4 intron-22-inversions, 3 large deletions, 2 missense mutations, 1 stop mutation, 1 insertion, 1 small deletion, 11 unknown) were recorded. 8 different factor VIII concentrates were given during inh development (5 plasma derived, 3 recombinant). Way of application (16 bolus infusion, 3 continuous infusion, 5 times both), infused amount until inh development (3000 IU to >1 mio IU), inh characteristic (15 HR, 9 LR), concomitant diseases and medication were registered. In conclusion it became obvious that inh in PTP are still a serious and underestimated problem in haemophilia treatment today. Our patient numbers are still too small to draw conclusions concerning given F VIII products or way of application. Secondly data showed that there is a variety of PTP definitions in Germany, referring to age of pat, number of ED and former change of product. A definition from the SSC of the ISTH for PTP would be helpful. The continuous use of the German register for drug side effects would make it easier to evaluate data in the future. A prospective, not product related study should be conducted.

Haemophilia A: Mutation Type Determines Risk of Inhibitor Formation

1995 ◽  
Vol 74 (06) ◽  
pp. 1402-1406 ◽  
Author(s):  
R Schwaab ◽  
H-H Brackmann ◽  
C Meyer ◽  
J Seehafer ◽  
M Kirchgesser ◽  
...  
Keyword(s):  
Factor Viii ◽  
Antibody Formation ◽  
Gene Mutations ◽  
Therapeutic Effects ◽  
Haemophilia A ◽  
Missense Mutations ◽  
Factor Viii Gene ◽  
Large Deletions ◽  
Intron 22 Inversion ◽  
First Time

SummaryThe formation of factor VIII antibodies is a major problem for replacement therapy of haemophilia A patients. Antibodies occur in 5-30% of patients with severe haemophilia A. The reason for antibody formation is still unknown. In this study we correlate for the first time different factor VIII gene mutations, stop- and missense mutations, large and small deletions and intrachromosomal intron 22 recombinations to antibody formation. A total of 364 patients with known inhibitor status of our institute, of the database, and of 3 studies representing intron-22-inversion data are included. The results show that the risk for developing factor VIII antibodies is strongly related to stop mutations, large deletions and intrachromosomal recombinations. A probable explanation could be the complete lack of endogenous circulating factor VIII protein in these cases. Other factors that might be important for the pathogenesis of inhibitor formation, e. g. the antenatal period, as well as possible therapeutic effects, are discussed.

POINT-MUTATION OF FACTOR VIII CODING SEQUENCES IN HAEMOPHILIA A

1987 ◽  
Author(s):  
R J Matthews ◽  
I R Peake ◽  
A L Bloom
Keyword(s):  
Factor Viii ◽  
Point Mutation ◽  
Cdna Probe ◽  
Factor Viii Inhibitor ◽  
Haemophilia A ◽  
Severe Haemophilia ◽  
Coding Region ◽  
Cdna Fragment ◽  
Coding Sequences ◽  
Restriction Sites

In order to study the molecular basis of haemophilia A, DNA from 26 haemophilia A patients (8 severe with inhibitors, 13 severe noninhibitors and 5 mild/moderate) was screened by the Southern blotting method with FVIII cDNA probe A (a i.7kb Kpnl cDNA fragment that spans exons 1 to 12) probe B (a 4.7kb EcoRI cDNA fragment that contains exons 14 to 25 and part of exon 26) probe C (a 1.8kb EcoRI cDNA fragment that contains the remainder of exon 26 and probe D (an Apal/EcoRI 783bp cDNA fragment that includes all of exons 22 to 25 and parts of exons 21 and 26). All cDNA probes were kindly provided by Genetics Institutes Inc.No large structural alterations of the FVIII gene were detected in any of the patients. However altered TaqI restriction sites within the coding regions of 3 patients were observed. DNA from patient 1 with severe haemophilia (VIIIAg < 0.1 u/dl, inhibitor negative) when probed with probe C showed a substitution of the normal 2.6kb TaqI and 4.5kb EcoRI fragments with novel 12kb TaqI and 11.5kb EcoRI fragments respectively. In addition he showed the normal 4kb Bglll fragment with probe C. A point mutation or small deletion (50bp is suspected to be present within exon 26.Patient 2 had severe haemophilia and a FVIII inhibitor of 12 units (Bethesda). DNA from patient 2 when probed with probe B revealed a novel 5.0kb TaqI fragment instead of the normal 2.2kb and 2.8kb fragments.The location of the altered Taq I restriction site within the coding region of exon 18 was confirmed with intragenomic probe pi 14.12 that includes exons 17and 18 (kindly provided by Genentech Inc.) A family study with this mutation specific fragment showed the patients sister and mother to be carriers.DNA from Patient 3 (severe haemophilia, factor VIII inhibitor 33 units) when probed independently with probes B and D revealed the absence of the normal 2.4kb and 1.4kb TaqI fragments and the generation of a novel 3.8kb TaqI fragment suggesting an alteration of the TaqI site within the coding region of exon 23.The detection of altered TaqI restriction sites in 3of our patients is further evidence that 'CG' dinucleotide sequences might be relative hot-spots for mutation when occurring within coding sequences of genes.

Factor IX Gene Analysis In 70 Unrelated Patients with Haemophilia B: Description of 13 New Mutations

1999 ◽  
Vol 82 (11) ◽  
pp. 1437-1442 ◽  
Author(s):  
M. C. Trzeciak ◽  
A. Durin ◽  
G. Pernod ◽  
V. Gay ◽  
C. Ménart ◽  
...  
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Factor Ix ◽  
Coagulation System ◽  
Molecular Defect ◽  
Missense Mutations ◽  
Nucleotide Substitutions ◽  
Haemophilia B ◽  
Gradient Gel Electrophoresis

SummarySeventy unrelated patients suffering from haemophilia B have been screened for determining the molecular defect and for evaluating the spectrum of factor IX mutations in the Rhône Alpes region in France. Most patients were characterized with respect to factor IX antigen and factor IX coagulant activity. We have used denaturing gradient gel electrophoresis to obtain a full scanning of the whole coding, promoter, and exon flanking sequences of the factor IX gene. This technique enabled us to determine the molecular defect in 68 out of 70 families (97%), and the mutation was further identified in the two last patients with a direct sequencing of the gene. A total of 2 complete gene deletions in patients with antifactor IX inhibitor, 6 small insertions/ deletions and 62 point mutations were found. Two of these nucleotide substitutions (Arg145His and Ala233Thr) were detected in 21 patients (30%) suggesting the existence of a local founder effect. Thirteen mutations were previously undescribed, including 7 missense mutations. The detection of mutations in patients affected with haemophilia B may shed some light in the structure-function relationship of factor IX molecule within the coagulation system.

Mutational spectrum and deep intronic variants in the factor VIII gene of haemophilia A patients

2016 ◽  
Vol 36 (S 02) ◽  
pp. S25-S28 ◽  
Author(s):  
J. Oldenburg ◽  
C. R. Müller ◽  
S. Rost ◽  
J. E. Bach
Keyword(s):  
Factor Viii ◽  
Genomic Sequence ◽  
Genetic Disorders ◽  
Point Mutations ◽  
Diagnostic Methods ◽  
Haemophilia A ◽  
Missense Mutations ◽  
Factor Viii Gene ◽  
Current Standard ◽  
Intronic Variants

SummaryHaemophilia A (HA) is caused by a broad spectrum of different mutation types in the factor VIII gene (F8). In our patient cohort of more than 2600 HA patients as well as in other published studies, the most frequent cause are missense mutations in different F8 exons or the recurrent intron 22 inversion. Some exons and several specific nucleotide positions represent hot spots for point mutations in the examined cohort. About 4 % of cases remain without mutation after routine HA diagnostic methods including inversion PCRs, Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Deep intronic mutations cannot be detected by current standard HA diagnostics but have been reported for several genetic disorders. However, next generation sequencing (NGS) of the whole genomic sequence of the F8 gene allows to identify deep intronic variants. Conclusion: In general, NGS provides an effective approach to screen for different HA causing mutation types in the F8 gene.

Prospective surveillance study of haemophilia A patients switching from moroctocog alfa or other factor VIII products to moroctocog alfa albumin-free cell culture (AF-CC) in usual care settings

2015 ◽  
Vol 114 (10) ◽  
pp. 676-684 ◽  
Author(s):  
Laszlo Nemes ◽  
Victor Jimenez-Yuste ◽  
Luminita Rusen ◽  
Ana Cid ◽  
Robert Charnigo ◽  
...  
Keyword(s):  
Factor Viii ◽  
Clinical Manifestations ◽  
Surveillance Study ◽  
Haemophilia A ◽  
Severe Haemophilia ◽  
Inhibitor Development ◽  
On Demand ◽  
Fviii Inhibitor ◽  
Increased Risk ◽  
Clinically Significant

SummaryThis prospective, open-label, postauthorisation safety surveillance study assessed clinically significant inhibitor development in patients with severe haemophilia A transitioning from moroctocog alfa or other factor VIII (FVIII) replacement products to reformulated moroctocog alfa (AF-CC). Males aged12 years with severe haemophilia A (FVIII:C) < 1 IU/dl), > 150 exposure days (EDs) to recombinant or plasma-derived FVIII products, and no detectable inhibitor at screening were enrolled. Primary end point was the incidence of clinically significant FVIII inhibitor development. Secondary end points included annualised bleeding rate (ABR), less-than-expected therapeutic effect (LETE), and FVIII recovery. Patients were assigned to one of two cohorts based on whether they were transitioning to moroctocog alfa (AF-CC) from moroctocog alfa (cohort 1; n=146) or from another recombinant or plasma-derived FVIII product (cohort 2; n=62). Mean number of EDs on study was 94 (range, 1–139). Six positive FVIII inhibitor results, as determined by local laboratories, were reported in four patients; none were confirmed by a central laboratory, no inhibitor-related clinical manifestations were reported, and all anti-FVIII antibody assays were negative. Median ABRs were 23.4 and 3.4 in patients categorised at baseline as following on-demand and prophylactic regimens, respectively; 86.5 % of bleeding episodes resolved after one infusion. LETE incidence was 0.06 % and 0.19 % in the on-demand and prophylaxis settings, respectively. FVIII recovery remained constant throughout the study. No new safety concerns were identified. This study found no increased risk of clinically significant FVIII inhibitor development in patients transitioning from moroctocog alfa or other FVIII replacement products to moroctocog alfa (AF-CC).

Characterisation of two novel large F8 deletions in patients with severe haemophilia A and factor VIII inhibitors

2011 ◽  
Vol 105 (02) ◽  
pp. 279-284 ◽  
Author(s):  
Rolf Marschalek ◽  
Johannes Oldenburg ◽  
Florian Oyen ◽  
Reinhard Schneppenheim ◽  
Lukas Roth
Keyword(s):  
Dna Repair ◽  
Factor Viii ◽  
Endogenous Retroviruses ◽  
Haemophilia A ◽  
Severe Haemophilia ◽  
Dna Double Strand Breaks ◽  
Long Terminal Repeats ◽  
Large Deletions ◽  
Specific Pcr ◽  
Exon 1

SummaryLarge deletions are found in approximately 5% of patients with severe haemophilia A, but only a few deletion breakpoints have been char-acterised precisely so far. In this study we characterised the deletion breakpoints of two patients with severe haemophilia A, large deletions and factor VIII (FVIII) inhibitors, and subsequently established deletion-specific assays for the identification of carriers. Patient 1 had a deletion of 37,410 bp comprising exon 1 and the F8promoter region, and a 5 bp homology (GGGCC) is present at the chromosomal fusion site. In patient 2, a deletion of 22,230 bp including parts of intron 25, exon 26 and 3‘-UTR was identified. No homologous repetitive elements were found at the breakpoints. However, both breakpoints were located within long terminal repeats of endogenous retroviruses and the DNA motif TTTAAA – known to be able to bend DNA molecules – was identified at the centromeric breakpoint. By deletion-specific PCR experiments we were able to identify a heterozygous state in mother 2 (carrier) while mother 1 presented only with wild-type alleles (non-carrier). Both deletions are most likely created by DNA double strand breaks and subsequent DNA repair by the non-homologous end joining DNA repair pathway (NHEJ). The exact identification of the deletion breakpoints provides a reliable diagnostic tool for carrier identification in affected families by means of a deletion-specific PCR.

Haemophilia A Genotype and Inhibitor Risk: Preliminary Results of a French Haemophilia Centre.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3982-3982
Author(s):  
Annie Borel-Derlon ◽  
Mounia Slaoui ◽  
Jean Marc Costa ◽  
Jean Maurice Lavergne
Keyword(s):  
Direct Sequencing ◽  
Single Point ◽  
Large Deletion ◽  
Severe Form ◽  
Haemophilia A ◽  
Missense Mutations ◽  
Severe Haemophilia ◽  
Mild Form ◽  
Preliminary Results ◽  
Intron 22 Inversion

Abstract Caen Haemophilia centre monitored 343 haemophilia A patients, 62 of them with a severe defect. We report here the preliminary results of genotypes and factor VIII (FVIII) inhibitors response for the first 59 and particularly for 15 patients with FVIII inhibitors. For severe haemophilia A, the first step is a long PCR for intron 22 inversion, then PCR for intron 1 inversion. The second step is a direct sequencing of the FVIII gene in severe form without inversion and in moderate or mild disease. 41% (16 / 39) of the patients with severe haemophilia A have an intron 22 inversion, the remainder patients have single point mutations (17), principally on exon 14, large deletion (1), small deletion (3) or splice mutations (3). In the severe form, 10 of these patients have developed inhibitors: 6 with intron 22 inversion, 3 non sense mutations on exons 14 or 18, 1 large deletion on exon 14 and 1 splice mutation on intron 22. In the moderate or mild form, 20 patients have missense mutations, 5 of them have developed inhibitors. The genotype of three patients has been previously reported to be associated with a high incidence of inhibitors in moderate and mild form: Arg 593 Cys by loss of tolerance to exogenous and endogenous FVIII (1), Arg 2150 His by reducing the binding of FVIII to von willebrand factor (2). For the other two patients, we identified two missense mutations: Val 2017 Ala, Tyr 1786 Ser. We are processing genotypes for each haemophilia A patients to evaluate the genotype - phenotype relationship and the FVIII inhibitor risk related to genetic and environmental factors.

Factor VIII Inhibitors in Haemophilia A Patients Who Are Considered as Previously Treated Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4105-4105
Author(s):  
Charis von Auer ◽  
Johannes Oldenburg ◽  
Manuela Krause ◽  
Wolfgang Miesbach ◽  
Inge Scharrer ◽  
...  
Keyword(s):  
Factor Viii ◽  
Treatment Modalities ◽  
Haemophilia A ◽  
Missense Mutations ◽  
Large Deletions ◽  
Concomitant Variables ◽  
Stop Mutation ◽  
Factor Viii Concentrates ◽  
Previously Treated Patients ◽  
Previously Treated

Abstract Previously treated patients (PTP), who have not developed an inhibitor (inh.) so far, are considered to be tolerant to factor VIII and at low risk for inh. development. Therefore inh. detection in a PTP should raise concerns about the concomitant variables such as product neo-antigenicity or way of application. To find out about the current situation regarding inh. development in PTP in Germany, we conduct a retrospective study. A questionnaire was sent to 87 haemophilia treating centers, so far 46 of them answered. 28 PTP-inh. developments during the last 5 years were reported. Age of the patient (median 35), severity of haemophilia A (18 severe, 2 moderate, 8 mild), exposuer days (median 30) and gene mutation (intron-22-inversions 31%, large deletions 13%, missense mutations 25%, stop mutation 6%, small deletion 13%) were recorded. 8 different factor VIII concentrates were given during inh. development (5 plasma derived, 3 recombinant). Way of application (16 times bolus infusion, 3 times continuous infusion, 5 times both), former change of product, infused amount until inh. development, inh. characteristic, concomitant diseases and medication were registered. In conclusion most of the patients were over 18 years old (17/28) when the inh. developed. Most of them had severe haemophilia A (18/28) and had at least one change of product before inh. detection. Our data are still too small to draw any conclusion concerning product neo-antigenicity or treatment modalities. Nevertheless it became obvious that inh. development in PTP is still a serious and underestimated problem in haemophilia treatment today. Secondly data showed that there is a variety of PTP definitions in Germany, referring to age of pat, number of exposure days and former change of product. An international definition for PTP would be helpful. A national haemophilia register would make it easier to evaluate data in the future and a prospective, not product related study should be conducted.

Relationship between Factor VIII Mutation Type and Inhibitor Development in a Cohort of Previously Untreated Patients Treated with Recombinant Factor VIII (Recombinate™)

2000 ◽  
Vol 83 (06) ◽  
pp. 844-848 ◽  
Author(s):  
Ian Williams ◽  
Gordon Bray ◽  
Ian Peake ◽  
Anne Goodeve ◽  
Keyword(s):  
Factor Viii ◽  
Point Mutations ◽  
Gene Mutations ◽  
Protein Translation ◽  
Recombinant Factor Viii ◽  
Missense Mutations ◽  
Coding Region ◽  
Gene Deletions ◽  
Reading Frame ◽  
Mutation Type

SummaryA cohort of 79 previously untreated patients (PUPs) with moderatesevere haemophilia A (baseline Factor VIII <2%) were enrolled in a study to evaluate the safety, efficacy and immunogenicity of recombinant factor VIII (r-FVIII, Recombinate™). Blood samples were obtained retrospectively from a total 55 PUPs who were investigated for the spectrum of FVIII gene mutations responsible for their haemophilia. FVIII gene inversion mutations were found in 27 (49%) patients. Two patients had partial gene deletions. The remaining 26 patients were then screened for mutations in the FVIII gene coding region using conformation sensitive gel electrophoresis. Point mutations were identified in 22 (85%) of the patients and 14 of these mutations were novel. Study subjects were monitored for the development of FVIII inhibitors throughout the study. A total of 23 of the 73 evaluable subjects (including one subject with a low inhibitor titer at baseline) demonstrated an inhibitor on one or more occasions; 11 (15%) were persistent. Inhibitors were detected in patients with partial gene deletions and inversions and in three of eight patients with missense mutations. No inhibitors were found in 11 patients with small insertions or deletions resulting in an alteration of the protein translation reading frame (frameshift mutations). The results corroborate the observation that mutation type is an important determinant of the propensity to develop inhibitory anti-FVIII antibody.

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